Hypoxia-associated circPRDM4 promotes immune escape via HIF-1α regulation of PD-L1 in hepatocellular carcinoma

Abstract

Background: Hypoxia is a hallmark of cancer, and is closely intertwined with tumor immune evasion. Circular RNAs (circRNAs) have been implicated in tumor response to immune checkpoint blockades. However, hypoxia-associated circRNAs that orchestrate the association between hypoxia and response to immunotherapy remain poorly understood. Here, we aimed to determine the roles of hypoxia-associated circRNAs in immune escape of hepatocellular carcinoma (HCC) cells. Methods: Differentially expressed hypoxia-associated circRNAs were determined using high-throughput sequencing technology. HCC patients treated with PD-1 blockade were enrolled to assess the clinical significance of circPRDM4. RT-qPCR, western blotting, flow cytometry, T cell-mediated tumor cell killing assay, and enzyme linked immunosorbent assay were used to investigate the roles of circPRDM4 in immune escape of HCC cells in vitro. Patient-derived xenograft mouse models and adoptive human tumor infiltrating lymphocyte-CD8+ T cell transfer were adopted to evaluate the effects of circPRDM4 in vivo. RNA pull-down, mass spectrometry, RNA immunoprecipitation, chromatin immunoprecipitation, chromatin isolation by RNA purification, dual-luciferase reporter assays, dot blotting, DNA in situ hybridization, and immunoprecipitation were utilized to examine the interaction between circPRDM4, HIF-1α, and CD274 promoter. Results: We identified circPRDM4 as a hypoxia-associated circRNA in HCC. circPRDM4 was upregulated in responders to PD-1 blockade and associated with therapeutic efficacy. In vitro and in vivo experiments showed that circPRDM4 induced PD-L1 expression and promoted CD8+ T cell-mediated immune escape under hypoxic conditions. Mechanistically, circPRDM4 acted as a scaffold to recruit HIF-1α onto CD274 promoter, and cemented their interaction, ultimately promoting the HIF-1α-mediated transactivation of PD-L1. Conclusions: These findings illustrated that circPRDM4 promoted immune escape of HCC cells by facilitating the recruitment of HIF-1α onto the promoter of CD274 under hypoxia, thereby inhibiting CD8+ T cell infiltration in the tumor microenvironment. This work may provide a novel prognostic biomarker and therapeutic candidate for HCC immunotherapy.